The present invention refers to the recombinant vaccine against canine visceral leishmaniasis containing the recombinant A2 protein and saponin, as an adjuvant, allowing the distinction between vaccinated and infected animals through conventional ELISA or immunofluorescence tests that employ antigens of promastigote forms of Leishmania. 
The many leishmaniasis constitute a group of parasitical diseases that clinically present themselves as cutaneous or mucocutaneous wounds, or in the form of a visceral infection. They occur due to the infection by a variety of protozoan species belonging to the genus Leishmania (World Health Organization, Program for the surveillance and control of leishmaniasis, who dot in slash emc slash diseases slash leish slash index dot html, 2005). The many leishmaniasis are endemic in about eighty-eight countries. Of these, seventy-two are developing countries, and in this group are included thirteen of the countries with the lowest development rate in the world. The visceral form occurs due to infections caused by the species Leishmania (Leishmania) donovani and L. (L) infantum in countries of Europe, Asia, Africa and the Middle East, and due to the specie L. (L) chagasi in Latin American countries (Desjeux, Comp. Immunol. Microbiol. Infect Dis. 27:305-318, 2004). Alterations in the functions of the spleen, the liver and the bone marrow are observed on infected patients, and the infection may become chronic, causing irregular long-lasting fever, hepatosplenomegaly, lymphadenopathy, anemia, leucopenia, oedema, progressive enfeeblement and weight-loss, and possibly causing death if treatment is not administered. Infected individuals may also remain asymptomatic, though 20% of the individuals in endemic regions develop the classic form of the disease. The symptoms are progressive, and complications deriving from the infection's evolution are responsible for the greater part of the deaths (Sundar & Rai, Clin. Diagn. Lab. Immunol. 9:951-958, 2002).
L. (L) chagasi has got vast geographic distribution in the Americas, being found in Brazil, Argentina, Colombia, Bolivia, El Salvador, Guatemala, Honduras, Mexico, Paraguay, and Venezuela. Species such as marsupials and skunks are well-known wild reservoirs of the parasite. In domestic environments, the dog is considered the main reservoir in the domestic transmission cycle of visceral leishmaniasis (VL), due to the high prevalence of the canine infection as compared to the human one. Infected dogs, even the asymptomatic ones, present a great quantity of parasites in the skin, facilitating the infection of the vector insect from this reservoir, and, consequently, the transmission of the disease to people (Tesh, Am. J. Trop. Med. Hyg. 52:287-292, 1995).
Canine VL treatment, whichever the medicine used, is not viable as a measure for the control of the disease, for it is costly. Moreover, treated and clinically cured dogs frequently display returns of the disease, remaining as infection sources for the vector, and it raises the chance of selecting lineages that are resistant to such medicines, with serious implications for human treatment (Gramiccia & Gradoni, Int. J. Parasitol. 35:1169-1180, 2005).
This fact, associated to the lethalness of human VL when not treated, has taken the World Health Organization (WHO) to profess the elimination of dogs when they are seropositive for Leishmania antigens, as a measure for Public Health organizations controlling the disease. In this way, Brazil's Health Ministry adopted such procedure. Therefore, one of the most used actions in VL control is the elimination of infected dogs, which are detected through serological diagnosis or by the presence of clinical symptoms. However, such procedures bring deep sadness and indignation to their owners, who, many times, prefer omitting the disease to the competent organizations until the animals are near their deaths, when they become important transmitters of the parasite (Tesh, Am. J. Trop. Med. Hyg. 52:287-292, 1995).
Most research on vaccine development is based on the identification of molecules of the parasite and in immunization protocols that can induce Th1 cellular immune response, an essential requirement for inducing protection to the disease. Among dogs, the resistance or susceptibility to the disease is, probably, also associated to the dichotomy of the Th1/Th2 response. The resistance is associated to high specific lymphoproliferative response and with positive delayed hypersensitivity reaction (DHR), besides low quantity of parasite-specific antibodies. Resistance to the infection and protection, among dogs, would be related to a high interferon-gamma (IFN-γ) and nitric oxide (NO) production, and to the leishmanicidal activity of the parasite-infected macrophages, which means a Th1 immune response profile. High levels of IgG1 antibodies would be related to susceptibility, while high levels of IgG2a would be associated to resistance (Moreno & Alvar, Trends Parasitol. 18:99-405, 2002; Molano et al., Vet. Immunol. Immunopathol. 92:1-13, 2003). Therefore, in studies which assess vaccine effectiveness against infection by Leishmania, IFN-γ and IgG2 (dogs) or IgG2a (mice) antibodies are used as Th1 response markers and resistance-inductor markers, interleukin-4, interleukin-10 and IgG2 (dogs) or IgG2a (mice) antibodies, on the other hand, are used as Th2 answer and susceptibility markers.
Vaccines against canine Leishmaniasis are hard to develop, and due to this are still rare. One canine vaccine is available, LEISHMUNE® vaccine. It uses as its vaccine active principle a purified antigenic complex, including proteins, that corresponds to the Fucose-Mannose Ligand (FML), present in the parasite's surface, according to Brazilian patent request PI 9302386-3 (composition containing fractions of Leishmania cells called fml antigen, “fucose-mannose ligand” or “fucose-mannose connecter”, use of the fml antigen and its subfractions and components for applications in immunodiagnosis specific to human and animal visceral leishmaniasis, for applications in vaccines and for treatment or immunotherapy against human and canine visceral leishmaniasis).
LEISHMUNE® vaccine's primary characteristic is the induction of humoural response. The vaccinated dog rapidly develops a response through the production of specific antibodies against the parasite. Many tests, presented by the mentioned vaccine's inventor and partners, show that the vaccine protects approximately 86% of the animals which received it when placed in endemic areas. These studies' results were questioned by the scientific community, as well as by the industry, since the control and the vaccinated animals were located in different cities. Another important failure of the mentioned test was the presence of dogs, in both groups, using insect-repellent-impregnated collars. The parasite is transmitted by an insect's sting, and if the contact with the vector insect is deterred by use of repellent collars the dog is not really exposed to the alleged natural challenge. Based on the above data, the described protection percentage is questionable. Therefore, other studies have been carried out and some are being carried out by request of the public health regulatory organization.
Particularly, in what pertains to public health, it is known that according to WHO regulations seropositive animals must be sacrificed. It is also known that this measure is adopted in Brazil. Once having received this vaccine, the animal will develop heavy response through antibodies specific to the parasite, becoming seropositive. The diagnosis professed by the public organizations is the serologic one, due to it being cheap and easily executable, thus capable of being applied to all regions of the country without further problems. The vaccinated dogs must be sacrificed.
The indistinction, by traditional methods, of infected animals from vaccinated ones, simply creates a great public health problem. The owners of vaccinated animals show the vaccine card and do not allow the animal to be sacrificed. Taking into account that for each one hundred animals which received the vaccine, about fourteen may become infected, leading to an increase in the amount of possible domestic leishmaniasis reservoirs. Another important issue is related to epidemiologic inquiries made by the Ministry of Health, which are an important form of identifying the evolution of the disease in different regions. This inquiry is partially based on the serologic results of the dogs. With the advent of LEISHMUNE® vaccine, vaccination the obtained results are not real, for seropositive animals may not be infected, but merely vaccinated. It is possible to distinguish between dogs that are seropositive due to vaccination or due to infection by exams that detect the parasite's presence, such as Polymerase Chain Reaction (PCR) or immunocytochemistry. However, to perform these tests, requires fine techniques and costly equipment and reagents, besides trained technicians, in order to guarantee the accuracy of the results. PCR is done in private clinics; its use in public health is hard to be implemented and of elevated financial cost.
In addition to this, LEISHMUNE® vaccine has got high production costs, arriving at the market at prices that make it impossible for the whole population to have access to it. It is known that Brazil has a large low income population, and these people do not have access to the mentioned product. As this is a disease that expands itself in many regions of the country, it is ever more important to adopt measures that hinder the continuity of the parasite's transmission, especially the infection of dogs that live in domestic or adjoining areas. The possible use of this product in public health campaigns would be costly, besides the aforementioned problems. LEISHMUNE® vaccine presents interesting immunologic characteristics, but goes against the control measures adopted for the current epidemic, deterring the sacrifice of seropositive dogs, interfering in the epidemiologic inquiries and also being of high cost for public health usage.
The A2 antigen has been identified, initially, in the specie L. (L) donovani, by Charest & Matlashewski (Mol. Cell. Biol. 14:2975-2984, 1994), by a library of amastigote forms of L. (L) donovani cDNA. Multiple copies of the A2 gene are grouped in the L. (L) donovani chromosome 22 (850 kb). These genes are maintained in the species L. (L) donovani, L. (L) infantum, L. (L) chagasi, L. (L) amazonensis and L. (L) mexicana (Ghedin et al., Clin. Diagn. Lab. Immunol. 4:30-535, 1997).
In previously conducted searches through patent databases, there were found patent applications related to the usage of the A2 antigen as a reagent for leishmaniasis vaccination, as described below. U.S. Pat. No. 5,733,778 states the nucleotide sequence of the A2 gene and claims the protection of its expression in microbial hosts. The VL9 L. (L) donovani string's A2 gene sequence is deposited in GenBank, as described below:
LOCUS S69693 2817 bp mRNA linear INV 26-MAR-2002ACCESSION S69693VERSION S69693.1 GL546453ORGANISM Leishmania (donovani) infantumEukaryota; Euglenozoa; Kinetoplastida; Trypanosomatidae;Leishmania.REMARK GenBank staff at the National Library of Medicine created thisFEATURES Location/Qualifierssource 1..2817/organism = “Leishmania donovani infantum”/mol_type = “mRNA”/strain = “Ethiopian LV9”/sub_species = “infantum”/db_xref = “taxon:5662”/dev_stage = “amastigote”gene 1..2817/gene = “A2”CDS 72.782/gene = “A2”/note = “Plasmodium falciparum S antigen homolog; Thissequence comes from FIG. 5A”/codon_start = 1/product = “stage-specific S antigen homolog”/protein id = “AAB30592.1”/db xref = “GI:546454”ORIGIN (SEQ ID NO: 1)   1gagctccccc agcgaccctc tcggcaacgc gagcgcccca gtccccccac gcacaacttt   61gaccgagcac aatgaagatc cgcagcgtgc gtccgcttgt ggtgttgctg gtgtgcgtcg  121cggcggtgct cgcactcagc gcctccgctg agccgcacaa ggcggccgtt gacgtcggcc  181cgctctccgt tggcccgcag tccgtcggcc cgctctctgt tggcccgcag gctgttggcc  241cgctctccgt tggcccgcag tccgtcggcc cgctctctgt tggcccgcag gctgttggcc  301cgctctctgt tggcccgcag tccgttggcc cgctctccgt tggcccgctc tccgttggcc  361cgcagtctgt tggcccgctc tccgttggct cgcagtccgt cggcccgctc tctgttggtc  421cgcagtccgt cggcccgctc tccgttggcc cgcaggctgt tggcccgctc tccgttggcc  481cgcagtccgt cggcccgctc tctgttggcc cgcaggctgt tggcccgctc tctgttggcc  541cgcagtccgt tggcccgctc tccgttggcc cgcagtctgt tggcccgctc tccgttggct  601cgcagtccgt cggcccgctc tctgttggtc cgcagtccgt cggcccgctc tccgttggcc  661cgcagtctgt cggcccgctc tccgttggcc cgcagtccgt cggcccgctc tccgttggtc  721cgcagtccgt tggcccgctc tccgttggcc cgcagtccgt tgacgtttct ccggtgtctt  781aaggctcggc gtccgctttc cggtgtgcgt aaagtatatg ccatgaggca tggtgacgag  841gcaaaccttg tcagcaatgt ggcattatcg tacccgtgca agagcaacag cagagctgag  901tgttcaggtg gccacagcac cacgctcctg tgacactccg tggggtgtgt gtgaccttgg  961ctgctgttgc caggcggatg aactgcgagg gccacagcag cgcaagtgcc gcttccaacc 1021ttgcgacttt cacgccacag acgcatagca gcgccctgcc tgtcgcggcg catgcgggca 1081agccatctag atgcgcctct ccacgacatg gccggaggcg gcagatgaag gcagcgaccc 1141cttttccccg gccacgacgc cgcgctgagg cgggccccac agcgcagaac tgcgagcgcg 1201gtgcgcgggc gctgtgacgc acagccggca cgcagcgtac cgcacgcaga cagtgcatgg 1261ggaggccgga ggagcaagag cggtggacgg gaacggcgcg aagcatgcgg cacgccctcg 1321atgtgcctgt gtgggctgat gaggcgcgga tgccggaagc gtggcgaggg catcccgagt 1381tgcaccgtcg agtcctccag gcccgaatgt ggcgagcctg cggggagcag attatgggat 1441gcggctgctc gaagcgaccg agggcgctga ccggaaggtg gcccacttcc tcctcgggcc 1501tgtgcggcat ccgccctcga tcgggagccc gaatggtggc cgcgcgggtg aaggcgtgcc 1561gcccacccgc gtctccgtgt ggcgccgctg ggggcaggtg cgctgtggct gtgtatgtgc 1621gctgatgtgc tgacttgttc gtggtgggct atgggcacgg tgaggggcga cgttggccct 1681tgctgacttc ctctgctttc ttattattct cagtgccccc gctggattgg gctgcatcgg 1741cggtctgtat cgcgcttgtc tctctcattt gacggctgcg cgcctcccgc ccctcccact 1801cgtgctgtgg gatggaggca cggccgggct ctgtgttgtg tgcaccgcgt gcaagaattc 1861agatgaggga ctgccgagcg agcagacaaa gcagcagcag caacaggaag gcaggcctga 1921gcacgttttc ttttctctct tgagactgcg gactacggga atcagagacg tcgtcagaga 1981cgcgcatccg cacccgcgcg ctatgcttcc tcgttctctc tcccgcccca ttctgtgcgc 2041ctgcctgtct gcgtgtcgcg agcgccgttg ccggcggtct ctctcccctc ccttcgcttc 2101tctcttgcaa gcgcttcctt tttcacagcc gaacgttgct gctcgcctgg aggccgttcc 2161ccctcttatc atctctgcat ttatttttac acgtgctttt gctttggctt cctgacgatg 2221ccggccacct caccgcggtg tcagggccca gcgcccactc tttgtgggca ggccaagtag 2281cctgcagcct gcccatgagc acggctgtgg actcttggtg ccagcggaca ggtgtgggct 2341ggcgctgtgc cggtgacacc aacggtcatg atgacgcttg gaccagctca ctgcggatca 2401tgccgacgat tcaacgaatg cgcgcatcca cctactgcct ttctgccttt gctgcgctgc 2461ggtggtgctg agcgtggtcc cggggcctag cctgcgctgt acgcagcggc attgcggtgg 2521gctgagcggc gccaggcggt gctggccggc cctgctgctt ggcatagccg tggcgtgcag 2581cagatgcgga tgggctgtgg ctgcgcatgc gtgtgtgcgt tgacttgttc gtggtgggcg 2641ggcacgtaaa cggcaaaatg cgctttggcg ttccggcgcc acgctccggc gctggtgcgg 2701tattcgaata cgcgcctgaa gaggtggcga ggaaaatggc acgaggcgca gagggaaaaa 2761acgaaaagtg caaagtgcgc aaaccgcgca gaaaatgcgg gaaaaacgaa aagtgca
U.S. Pat. No. 5,780,591, WO 95/06729, EP 0716697 and MX PA03008832 report the amino acid sequence of the A2 protein. A2 is composed of a sequence of ten amino acids, repeated from forty to ninety times, depending on the “A2 family” gene that encodes it (Charest & Matlashewski, Mol. Cell. Biol. 14:2975-2984, 1994; Zhang et al., Mol. Biochem. Parasitol. 78:79-90, 1996), as shown below (SEQ ID NO: 2):
MKIRSVRPLWLLVCVAAVLALSASAEPHKAAVDVGPLSVGPQSVGPLSVG PQAVGPLSVGPQSVGPLSVGPQAVGPLSVGPQSVGPLSVGPLSVGPQSVG PLSVGSQSVGPLSVGPQSVGPLSVGPQAVGPLSVGPQSVGPLSVGPQAVG PLSVGPQSVGPLSVGPQSVGPLSVGSQSVGPLSVGPQSVGPLSVGPQSVG PLSVGPQSVGPLSVGPQSVGPLSVGPQSVDVSPVS
U.S. Pat. No. 5,780,591, besides describing the A2 native protein, i.e., descrybing how it is found in the parasite, claims its possible use as a vaccine or as a diagnosis antigen. U.S. Pat. No. 5,733,778 describes the DNA sequence of the A2 gene and its bacterial expression. U.S. Pat. No. 6,133,017 describes the obtainment of attenuated parasites (Leishmania donovani) by the deletion of the A2 gene, as well as their utilization as attenuated vaccines. WO 95/06729, EP 0716697 and MX PA03008832 are related one to another and claim the utilization of the A2 antigen in the form of a recombinant protein, DNA or attenuated parasites as a vaccine. In Brazil, PI0208532 (WO 02/078735) was registered on INPI (National Institute of Industrial Property), under the generic title of “Vacina Contra Leishmania” (i.e., Vaccine Against Leishmania), which describes the invention of a DNA vaccine whose antigenic component is the A2 antigen, and also describes the processes for administering this DNA vaccine that induces immune response to Leishmania infection in the host to which it is administered.
The experimental evidences that supported the protection requests for the vaccines above, however, consist essentially of two vaccination studies (Ghosh et al., Vaccine 19:3169-3178, 2001; Ghosh et al., Vaccine 20:59-66, 2001). In these studies experimental models (mice) were assessed, making use of the A2 antigen under the recombinant protein associated to Cornybacterium parvum, as an adjuvant, or in DNA form with the plasmid pcDNA3/E6. According to those studies, animals immunised with the A2 antigen and challenged with L. donovani have presented significant reduction of the parasite load in the liver and high production of IFN-γ and IgG2a antibodies specific to the A2 protein. Even though the vaccination with A2 DNA granted protection, the protection was more significant when the DNA was associated to the plasmid pcDNA3/E6. Albeit necessary for the validation of a vaccine formulation's efficacy, the assessment step in many experimental models cannot be considered conclusive, even when it presents positive results.
The development of a vaccine that is effective in protecting the dog and, consequently, in lowering the chances of transmission to humans, would be of great relevance for the control of leishmaniasis. In Brazil, the Health and Agriculture Ministries, according to an edict that is available to public consultation (www dot mapa dot gov dot br), profess that this vaccine, once applied to the dogs, must be able to induce an immunologic response effective in reducing tissue parasitism and the transmission of the parasite to the vector insect. This can be verified through xenodiagnosis, polymerase chain reaction (PCR) or immunohistochemistry. In addition to this, the vaccine must allow the serologic distinction between vaccinated and infected dogs while employing low-cost laboratorial methods that are available to the public network, thus not encumbering the country's Health System. Therefore, the adoption of a vaccine as a new control measure must not interfere with the current control measures.